The vacuolar ATPase is the primary proton pump in the endomembrane system of eucaryotic cells. The ATPase serves two functions. First, it generates an electrochemical gradient across the membrane that can be used to drive the transport of other molecules. Second, the ATPase can generate an acidic environment within a cellular compartment. Understanding the vacuolar ATPase is likely to have tremendous medical importance because of its diverse physiological roles. For example, vacuolar ATPase has been reported to be involved in growth of tumors, development of Alzheimer's, infection by mycobacteria, and the uptake of viruses. This proposal outlines experiments to examine the structure of the vacuolar ATPase and to investigate its physiological role in the cell. The binding site of a family of specific inhibitors, the bafilomycins and concanamycins, will also be investigated. Neurospora crassa will be used as the experimental organism because it is particularly amenable to both biochemical experiments and to genetic manipulation. Our previous work has shown that the vacuolar ATPase is a large and complex enzyme. In electron micrographs it appears as a "ball and stalk" projecting from the membrane. The peripheral sector, named Vo, is composed of at least 6 different polypeptides. The membranes sector, named Vo, contains at least 4 different polypeptides. Thus far genes that encode six of these subunits in N. crassa have been isolated. In addition, a method has been developed for generating strains with null mutations in genes encoding ATPase subunits. In this proposal the specific aims are: (1) to characterize genes that encode the 50, 17, and 14 kDa subunits of the Vo sector; (2) to purify the 20 kDa polypeptide of the Vo sector and isolate the gene encoding it; (3) to develop new methods to purify the enzyme in order to define more completely the subunit composition; (4) to use biochemical and genetic experiments to identify the binding site(s) for bafilomycin and concanamycin; and (5) to investigate the physiological role of the enzyme by constructing strains with altered levels of vacuolar ATP activity.